The phospholipase A2 (PLA2) subclass of lipolytic enzymes plays a central role in membrane phospholipid homeostasis, phospholipid remodeling, signal transduction processes, and general phospholipid metabolism. This enzyme also catalyzes the release of arachidonic acid, the essential first step in the biosynthesis of the eicosanoids, including prostaglandins and leukotrienes that are important for a large number of inflammatory and other diseases. PLA2 is not a single enzyme but rather a superfamily of enzymes that currently includes over ten groups. These enzymes vary in size from 14 to 85 kDa, they include both Ca2+ dependent and independent forms, and some are secreted while others act intracellularly. To understand how this diverse array of enzymes act and interact in cells to accomplish all of their tasks will require a detailed understanding of how each enzyme works. This will require the isolation or expression of sufficient quantities of each enzyme to allow detailed kinetic and physical studies to be carried out. In this grant, we propose a comprehensive study of key aspects of representatives of four groups of this enzyme superfamily. We will complete the characterization of the cobra venom Group IA PLA2 by identifying its PC activator site. The Group IA PLA2 will also be studied using a new technique that uses phospholipid "bicelles" to study the structure and orientation of enzymes at lipid-water interfaces. We will characterize the PIP2 binding characteristics of the Group IV cytosolic PLA2. We will also express and characterize two recently discovered isoforms of the enzyme, i.e. the Group IVB and IVC also known as cPLA2beta and cPLA2gamma. We have recently cloned and expressed the novel Group V sPLA2. We now propose to carry out detailed kinetic and binding studies of the enzyme and to use various mutants of the enzyme to determine the amino acid residues involved in catalysis. We will compare the characteristics of the Group V enzyme with the Group IIA enzyme since it appears that these two enzymes play very similar roles in cells. The final enzyme that we propose to study is the Group VI Ca2+-independent PLA2. We will mutate this enzyme to determine the catalytically important amino acid residues. We will also use mutagenesis to study the role of the enzyme's ankyrin repeats in catalysis.